1. Field of Invention
This invention generally relates to the field of molecular medicine and in particular to a novel set of heat-shock protein-binding proteins, and to polynucleotides encoding them, useful in the regulation of physiological events in which one or more 70 kiloDalton heat-shock proteins (Hsp70) are involved, such as normal development, cellular stress responses, heart disease, and cancer.
2. Description of the Related Art
Practically all organisms respond to heat by inducing the synthesis of a group of proteins called the heat-shock proteins. Although the details of this response vary among organisms, the involvement of Hsp70 and Hsp90 gene families is known to be highly conserved. More recently, it has come to be known that heat shock proteins can be induced by a variety of stress-related stimuli besides heat: anoxia, ethanol and certain heavy metal ions also stimulate increased expression and activity by these proteins. Hence, such proteins commonly are more broadly referred to by those in the art as heat stress or, simply, stress proteins.
Interestingly, stress proteins also are present within cells under non-stressful conditions (i.e. under normal physiological conditions). Genetic studies in bacteria and lower eukaryotes have demonstrated that Hsp70 is essential for growth at either high or normal temperatures, indicating a crucial role in normal cellular physiology. See generally S. Lindquist and E. A. Craig, The Heat Shock Proteins, Annual Revue of Genetics. 22:631-77 (1988).
Particular attention has been focused on Hsp70, a member of a multigene family whose genes are expressed under a wide variety of environmental conditions and are found in all cells. As shown schematically in FIG. 1, Hsp70 and related proteins (such as Hsp72, Hsc70, and Grp78) contain an ATPase domain, a substrate binding domain, and a coupling domain. S. Lindquist and E. A. Craig, Annual Revue of Genetics. 22:631-77 (1988).
In terms of function, studies have shown that Hsp70 plays a role in DNA replication, transport of proteins across membranes, binding of proteins to the endoplasmic reticulum, and uncoating clathrin coated vesicles. S. Lindquist and E. A. Craig, Annual Revue of Genetics. 22:631-77 (1988). Furthermore, Hsp70 is known to associate with nonsterified fatty acids, palmitic acid, stearic acid, and myristic acid and to be involved in signal transduction pathways in the cytoplasm. Hohfeld, Jorg, et al., Hip, a Novel Cochaperone Involved in the Eukaryotic Hsc70/Hsp40 Reaction Cycle. Cell vol. 83, 589-598 (Nov. 17, 1995).
Of these functions, perhaps the best studied has been the role of Hsp70 as a “chaperone,” a protein that stabilizes other proteins against aggregation and that mediates the folding of newly translated polypeptides in the cytosol and organelles. Proper functioning of Hsp70 as a protein chaperone is dependent on its bound nucleotide state. Specifically, the ATP form of Hsp70 binds substrate very poorly and therefore must be converted to the ADP form before the misfolded protein can bind. Then, the high affinity of Hsp70 for ATP is utilized to “power” the protein folding and other functions of Hsp70, as much energy is generated by the hydrolysis of bound ATP.
The search for regulators of Hsp70 chaperone function has revealed regulatory factors that form complexes with Hsp70 and assist in determining those substrates with which Hsp70 can associate. For example, the DNAJ-like proteins bind protein substrates exhibiting secondary and tertiary structure but have very low affinity for polypeptides in unfolded conformations. On the other hand, Hsp70 proteins bind unfolded proteins best. Thus, by forming a complex with DNAJ-like protein, Hsp70 proteins can bind with many other proteins of varying conformation. Cyr, D. M. et al., DnaJ-like Proteins: Molecular Chaperones and Specific Regulators of Hsp70. TIBS 19 (April, 1994).
Other factors can regulate the substrate binding stability or ATPase activity of Hsp70. Hsp40 stimulates the ATPase of Hsp70 and therefore results in production of the ADP form of Hsp70, which facilitates binding to substrate. Another Hsp70 regulator, the Hip co-chaperone protein, binds to the ATPase domain of Hsp70, thereby promoting the assembly of chaperone complexes and prolonging the time window during which a Hsp70 protein can interact stably with unfolded polypeptides. Hohfeld, Jorg, et al., Hip, a Novel Cochaperone Involved in the Eukaryotic Hsc70/Hsp40 Reaction Cycle. Cell vol. 83, 589-598 (Nov. 17, 1995). Similarly, a regulator named Hop modulates the binding of Hsp70 to Hsp90, thereby stimulating Hsp70-mediated refolding of a denatured protein. Johnson, B. D., et al., Hop Modulates Hsp70/Hsp90 Interactions in Protein Folding. JBC 273:6, pp. 3679-3686 (Feb. 6, 1998).
A potential regulator of Hsp70 is a 16-kDa protein that is a member of the Nm23/nucleoside diphosphate kinase family. This regulator monomerized Hsc70 (a protein closely related to Hsp70) and assisted in releasing Hsc70 from bound substrate. Leung, S. M. and L. E. Hightower, A 16-kDa Protein Functions as a New Regulatory Protein for Hsc70 Molecular Chaperone and Is Identified as a Member of the Nm23/Nucleoside Diphosphate Kinase Family. JBC 272:5, pp. 2607-2614 (Jan. 31, 1997). Also, the cysteine string protein, which is a secretory vesicle protein, and auxilin, a clathrin-associated protein, can both activate Hsc70 ATPase activity. Chamberland, L. H. and R. D. Burgoyne, Activation of the ATPase activity of heat-shock proteins Hsc70/Hsp70 by cysteine-string protein. Biochem. J. 322, pp. 853-858 (1997); Braun, J. E. A., et al., The Cystein String Secretory Vesicle Protein Activates Hsc70 ATPase. JBC 271:42, pp.25989-25993 (Oct. 18, 1996); Jiang, R. F. et al., Interaction of Auxilin with the Molecular Chaperone, Hsc70. JBC 272:10, pp. 6141-6145 (Mar. 7, 1997).
Still other regulators of Hsp70 inhibit Hsp70-mediated refolding. The RAP/HAP46 proteins, which inhibit binding of misfolded proteins to Hsp70, and BAG-1, which causes the release of ADP from Hsp70, both down-regulate Hsp70 activity. Zeiner, M. et al., Mammalian protein RAP46: an interaction partner and modulator of 70 dDa heat shock proteins. EMBO J. 16:18, pp. 5483-5490 (1997); Takayama, S. et al., BAG-1 modulates the chaperone activity of Hsp70/Hsc70. EMBO J. 16:16, pp. 4887-4896 (1997).
Despite the fact that regulators of Hsp70 protein binding have been discovered and characterized, the functional regulation of Hsp70 is not yet understood. Moreover, the ability to directly abrogate or eliminate Hsp70 ATPase activity through a selectively binding protein has not previously been known. Therefore, the discovery and isolation of polynucleotides encoding two isoforms of a human heat-shock protein binding protein (HspBP-1 and HspBP-2), is desirable because they provide a means to investigate the effects of heat shock-protein regulation. Such regulation may have consequences in physiological pathways or conditions in which Hsp70 is known to be involved, such as development, apoptosis, cellular stress, heart disease, and cancer.